DNA damage recognition in linear and supercoiled DNA

我们细胞中的特殊蛋白质已经进化到通过识别和修复DNA中的损伤来维持基因组的完整性，这些损伤是由环境污染物、太阳紫外线或DNA复制时引入的错配造成的。这些蛋白质需要快速扫描DNA，但在潜在的“麻烦点”放慢速度，以识别损伤并开始修复。由于细胞内的DNA是环状和超螺旋的(展开或过度缠绕)，这一过程变得复杂起来。该项目旨在揭示DNA环和超螺旋如何影响DNA修复蛋白与受损DNA的相互作用。这些研究中的发现和开发的技术将为理解其他蛋白质，如那些调节基因表达的蛋白质，如何与超螺旋DNA接触打开大门。该项目将产生教育影响，为不同的本科生和研究生提供参与跨学科前沿研究的机会；开发生物物理学的跨学科本科课程；与高中教师合作开发物理和生物学之间的教学模块；以及在夏季在他们的实验室接待高中生。公共和外展活动将包括每周一次的“周六早间科学”，邀请高中生谈论科学。缺乏对DNA损伤识别的分子水平的理解，这在很大程度上是因为在与询问和识别相关的微秒到毫秒时间尺度上测量蛋白质-DNA构象动力学是困难的，因此，很少。有几条证据表明，损伤感测蛋白可以感知局部DNA波动的差异，以及区分受损和未受损部位的变形能力。然而，表征DNA的动态和灵活性一直是该领域的一大挑战。更少见的是关于DNA拓扑(环状和超螺旋)--跨越生命领域的保守--如何影响这些动态的研究。使用创新的荧光方法，包括激光温度跳跃、基于荧光寿命的FRET和荧光相关光谱，该项目将建立在PI实验室之前的研究基础上，这些研究揭示了蛋白质-DNA构象动力学在通过核苷酸切除修复途径中的干皮病C蛋白的酵母同源物Rad4进行损伤识别的过程中。对错配修复途径中MutS蛋白错配识别的研究将测量不同错配的DNA动力学，可视化MutS如何与错配位点接触，并确定不同错配的错配询问/识别率是否与MutS介导的修复效率相关。利用可控超螺旋度设计的DNA小环将被用来研究环状和超螺旋对DNA涨落的影响，以及这对Rad4/MutS损伤识别的热力学和动力学的影响。这些研究的极高的灵敏度和时间分辨率将使测量RAD4/MutS介导的不匹配位置的扭结、解离、核苷酸翻转动力学形成识别复合体，阐明当DNA超螺旋时这些动力学是如何改变的，并为内在DNA波动如何停滞并向损伤感知蛋白质传递“求救信号”提供答案。该项目由遗传机制和生物科学局分子和细胞生物科学部的分子生物物理学项目共同资助。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的审查标准进行评估，被认为值得支持。

Special proteins in our cells have evolved to maintain genome integrity by recognizing and repairing damage in DNA caused by environmental pollutants, UV-rays from the sun, or mismatches introduced when DNA is replicated. These proteins need to rapidly scan DNA and yet slow down at potential “trouble spots” to recognize damage to begin repair. This process is complicated by the fact that DNA inside cells is looped and supercoiled (unwound or overwound). This project aims to uncover how DNA looping and supercoiling impact the interactions of DNA repair proteins with damaged DNA. The findings and the technologies developed in these studies will open doors for understanding how other proteins, such as those that regulate gene expression, engage with supercoiled DNA. The project will have educational impacts by providing opportunities for diverse undergraduate and graduate students to take part in cross-disciplinary cutting-edge research; developing an interdisciplinary undergraduate program in biophysics; engaging with high school teachers to develop teaching modules at the interface between physics and biology; and hosting high school students in their laboratories in the summer. Public and outreach activities will include a weekly “Saturday Morning Science” to bring in high school students to talk about science. A molecular-level understanding of DNA damage recognition is lacking, in large part because measurements of protein-DNA conformational dynamics on micro-to-milliseconds timescales relevant for interrogation and recognition are difficult and, thus, rare. Several lines of evidence suggest that damage sensing proteins sense differences in local DNA fluctuations and deformability to distinguish damaged from undamaged sites. Characterizing DNA dynamics and flexibility, however, has been a major challenge in the field. Even rarer are studies on how DNA topology (looping and supercoiling) – conserved across the domains of life – influence these dynamics. Using innovative fluorescence approaches that include laser temperature-jump, fluorescence lifetime-based FRET, and fluorescence correlation spectroscopy, this project will build on previous studies in the PI’s laboratory that unveiled protein-DNA conformational dynamics en route to damage recognition by Rad4, yeast ortholog of xeroderma pigmentosum C protein in the nucleotide excision repair pathway. Studies for mismatch recognition by MutS protein in the mismatch repair pathway will measure DNA dynamics for different mismatches, visualize how MutS engages with the mismatched sites, and ascertain whether mismatch interrogation/recognition rates for different mismatches correlate with MutS-mediated repair efficiencies. DNA minicircles designed with controlled superhelicity will be used to investigate the effect of looping and supercoiling on DNA fluctuations and what impact that has on the thermodynamics and kinetics of damage recognition by Rad4/MutS. The exquisitely high sensitivity and temporal resolution of these studies will enable measurements of Rad4/MutS-mediated kinking, unwinding, nucleotide-flipping kinetics at mismatched sites to form the recognition complex, shed light on how these dynamics are altered when DNA is supercoiled, and provide answers to how intrinsic DNA fluctuations stall and communicate “distress signals” to damage sensing proteins.This project is co-funded by the Genetic Mechanisms and the Molecular Biophysics Programs in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.